Lovastatin, pravastatin, simvastatin, mevastatin, atorvastatin and derivatives and analogs thereof are examples of known as HMG-CoA reductase inhibitors which are used as antihypercholesterolemic agents. The majority of them are produced biotechnologically by fermentation using microorganisms of different species identified as species belonging to Aspergillus, Monascus, Nocardia, Amycolat psis, Mucor or Penicillium genus, some are obtained by treating the fermentation products using the methods of chemical synthesis, thus leading to semi-synthetic substances, or they are the products of total chemical synthesis.
The present invention relates to a new industrial process for isolation and/or of HMG-CoA reductase inhibitors via salts thereof with specific amines. The invention enables a user to obtain the pure amine salts of HMG-CoA reductase inhibitors from the fermentation broth in case the substances are produced by biotechnological (microbiological) processes, or from the reaction mixture in case the substances are produced by semisynthetic or total chemical synthesis. The step of forming salts with amine may be one of the steps in the process for isolation and/or purification of HMG-CoA reductase inhibitors or precursor substances thereof. The amines described in the present specification are very useful for the formation of salts in the composition of media in processes for biotechnological modification of HMG-CoA reductase inhibitors or precursors thereof. The salts thus formed may be used as the starting substances or intermediates for the preparation of semisynthetic derivatives and analogs thereof, or by employing simple techniques known from the literature, if required, to be converted into the pharmaceutically acceptable salts and lactones, respectively.
The processes for the isolation and purification of antihypercholesterolemic agents known from patent and technical literature include different combinations of extraction, chromatography, lactonization and crystallization methods. Some of them additionally include the isolation and purification via different salts. In U.S. Pat. Nos. 9,342,767 and 4,319,039, the ammonium salt of lovastatin (in the carboxylate form) is isolated directly from the organic phase which has been extracted from the fermentation medium. In the same patent the preparation of ethylenediamine, tetramethylammonium, potassium and N-methylglucamine salts as well as the salts of different amino acids such as L-lysine, L-arginine and L-ornithine is also described. The aforementioned salts are prepared from the already purified substance and the option for their use in the process of isolation or purification is not mentioned. GB 2055100A also describes the formation of the sodium and calcium salts of lovastatin, which comprises the extraction in methanol, two steps of preparative liquid reverse-phase chromatography, crystallization from methanol and recrystallization from ethanol, and the conversion into the salt using an aqueous solution of sodium or calcium hydroxide. However, without including various chromatography methods, the methods described do not yield a product of the purity comparable to the product obtained by using the present invention. U.S. Pat. No. 4,346,227 discloses a process for the preparation of the sodium salt of pravastatin, wherein chromatographic techniques are also used but the final product is obtained only after lyophilization which is not an economical process in a large scale production operations. EP 65,835 discloses the preparation of the L-ornithine and t-octylamine salts of tetrahydro-M4 or tetrahydro-IsoM-4 (wherein M4 denotes a specific HMG-CoA reductase inhibitor, M4 and IsoM-4 representing the isomers hydroxylated at 6- and 3-biphenyl ring position, respectively, and xe2x80x9ctetrahydroxe2x80x9d means that the condensed biphenyl ring system is fully hydrogenated) as final products, that is from the respectively purified sodium salts thereof, but not as intermediates via which the isolation would be carried out. Other salts of tetrahydro-M-4 or IsoM:4 with ammonia, an amino acid or an organic amine are also contemplated as final products, including octylamine, 2-ethylhexylamine, benzylamine, a-methyl-benzylamine, phenethylamine, dibenzylamine, N-methylbenzylamine, N,N-dimethylbenzylamine, N,N-diethylbenzylamine, N-ethyl-N-methylbenzylamine, tribenzylamine, cyclopentylamine, cyclohexylamine cycloheptylamine, N-methylcyclopentylamine, N-ethylcyclohexylamine, N-ethylcycloheptylamine, dicyclohexylamine, N,N-dimethylcyclopentylamine, N,N-dimethylcyclohexylamine, N,N-diethylcycloheptylamine, pyrrolidine, N-methylpyrrolidine, piperidine, N-methylpiperidine and morpholine. GB 2073199A also discloses the preparation of different salts of HMG-CoA reductase inhibitors from the already isolated substance in the lactone form. U.S. Pat. Nos. 5,763,653 and 5,763,646 disclose the preparation of the cyclopropylamine and n-butylamine amides of lovastatin and their use in a process of chemical semisynthesis of simvastatin. U.S. Pat. No.5,403,860 discloses, as final products, amine salts of octahydronaphthalene oxime derivatives of HMG-CoA inhibitors, the derivatives deriving from ML-236A, ML-236B, MB-530A and MB-530B. As final amine salts, t-octylamine, dibenzylamine, dicyclohexylamine, morpholine, D-phenylglycine alkylester and D-glucosamine salts are mentioned.
In industry there exists a constant need for rationalization of the production and shortening of the production processes as well as for the use of least expensive starting raw materials or intermediate substances. To date the isolation of the final products in the case of HMG-CoA reductase inhibitors has been a multi-stage process wherein each step adds its share to the losses resulting in the final yield rarely greater than 60%. In addition, a product in the lactone form or lactone converted into the sodium salt is used as the starting substance in the process of semisynthesis (erg. in a process for preparing simvastatin) or biochemical conversion (e.g. in a process for preparing pravastatin). The preparation of lactone is one of the least economical steps in the production of HMG-CoA reductase inhibitors since losses in the course of the conversion from the acid into the lactone form and optionally further into the salts are greater than 20%. Therefore, there is a constant need for the starting substances and/or the intermediate substances which would be sufficiently pure, with small losses during their conversion, low costs, and the preparation per se should be technologically simple.
In our developmental and research work we have surprisingly found that HMG-CoA reductase inhibitors form the salts with certain amines which crystallize from mother liquor once they are formed. It has surprisingly been found that crystals of the amine salt of the desired HMG-CoA reductase inhibitor of high purity may be obtained from the liquors containing a large number of impurities and undesired HMG-CoA reductase inhibitor analogs. Contrary to the statements from U.S. Pat. No. 5,403,860 that lower yields are obtained when using the salts of HMG-CoA reductase inhibitor as starting or intermediate substances in a process for preparing the substances (Ia) mentioned below, we have surprisingly found that, when using the amine salts of HMG-CoA reductase inhibitors according to the present invention, the yields and the purity of the prepared HMG-CoA reductase inhibitors are equal to or greater than when using the HMG-CoA reductasel inhibitors in the lactone form.
Exemplified Formula Ia 
R1: CH3, CH2OH, OH
a and b: both may be double bonds, one of them may be a single bond, both may be single bonds
Furthermore, we surprisingly discovered that in processes for the biotechnological modification of HMG-CoA reductase inhibitors the formation of amine salts of HMG-CoA reductase inhibitors in the medium which derives from the fermentation liquor provides, in comparison with the mere metal salts as described in publicly accessible literature, an efficient means for the isolation and/or purification of HMG-CoA reductase inhibitors by means of simple crystallization. The amines which are described in the present specification and which readily form salts with HMG-CoA reductase inhibitors are thus particularly suitable as auxiliary materials or processing aids for the isolation and/or purification of HMG-CoA reductase inhibitors. Furthermore, they can be excellently used as starting materials or intermediates of semisynthetic preparation or biotechnological modification of HMG-CoA reductase inhibitors and, furthermore, for the conversion into pharmaceutically acceptable salts or into the lactone form of the respective HMG-CoA reductase inhibitors. Accordingly, the novel amine salts of HMG-CoA reductase inhibitors of the present invention are also highly valuable as such.
The present invention provides:
a) the novel salts of HMG-CoA reductase inhibitors with organic amines, wherein those specific salts are excluded which are disclosed in the prior art, but in different contexts as mentioned above,
b) a process for the preparation of salts of HMG-CoA reductase inhibitors with amines,
c) a use of salts of HMG-CoA reductase inhibitors with amines as processing aids or starting substances or intermediate substances in various processes,
d) a process for the preparation of the pure HMG-CoA reductase inhibitors from/via amine salts thereof,
e) a process for the semisynthetic preparation of HMG-CoA reductase inhibitors, wherein the amine salts of HMG-CoA reductase inhibitors are used as the starting substances,
f) a process for the biotechnological modification of HMG-CoA reductase inhibitors, wherein one of the components of the medium is the amine salt of HMG-CoA reductase inhibitors,
g) a process for the conversion of the amine salts of HMG-CoA reductase inhibitors into the pharmaceutically acceptable salts of HMG-CoA reductase inhibitors, and
h) a process for the conversion of the amine salts of HMG-CoA reductase inhibitors into HMG-CoA reductase inhibitors in the lactone form.
The amine which is used according to the present invention for the formation of the salts with a HMG-CoA reductase inhibitor is selected from the group consisting of organic amines of the following formulae I and II: 
wherein:
a1) R1, R2, R3 and R4 independently denote:
a hydrogen atom
a straight or a branched alkyl group having 1 to 8 carbon atoms, or
a cycloalkyl group having 3 to 8 carbon atoms, or
an arylalkyl group wherein the alkyl group is methyl or ethyl and the aryl group is phenyl, which is optionally substituted by an N-alkyl or N,N-dialkyl group wherein the alkyl group is alkyl having 1 to 4 carbon atoms, or
an arylalkyl group which is optionally substituted by one or more substituents,
a hydroxyalkyl group having 2 to 4 carbon atoms, or
an aminoalkyl group having 2 to 4 carbon atoms, which are optionally substituted by an N-alkyl or N,N-dialkyl group wherein the alkyl group is alkyl having 1 to 4 carbon atoms;
X denotes a hydrogen atom, a hydroxyl group, a halogen or a methyl group;
m and n independently denote an integer from 0 to 5; or
a2) NR1R2 or NR3R4 denote a heterocyclic ring having 3 to 7 methylene groups, one of these groups being optionally substituted by an oxygen or a sulphur atom or an imine group; X, m and n are as defined above; 
wherein:
b1) Rxe2x80x21, Rxe2x80x22, and Rxe2x80x23 are the same or different and denote hydrogen, alkyl, alkenyl, amino- or hydroxy- or alkoxy-substituted alkyl or alkenyl, or substituted amino-substituted alkyl or alkenyl, provided that Rxe2x80x21, Rxe2x80x22, and Rxe2x80x23 are not hydrogen at the same time; or
b2) Rxe2x80x21 and Rxe2x80x22, and optionally Rxe2x80x23, together with the nitrogen atom form an optionally substituted heterocyclic ring system including the nitrogen atom as a ring member, and optionally including an additional hetero atom, and if Rxe2x80x23 is not part of the ring system it is independently selected from hydrogen, alkyl, alkenyl, amino- or hydroxy- or alkoxy-substituted alkyl, or substituted amino-substituted alkyl; or
b3) Rxe2x80x21 is an optionally substituted cyclic group of general formula III,
Rxe2x80x2xe2x80x94(CHRxe2x80x24)mxe2x80x94xe2x80x83xe2x80x83III
xe2x80x83wherein m is zero or an integer from 1 to 5, Rxe2x80x2 is optionally substituted aliphatic hydrocarbon cyclic system having 3 to 8 carbon atoms in the ring, Rxe2x80x24 is hydrogen, or alkyl, amino- or hydroxy- or alkoxy-substituted alkyl, or substituted amino-substituted alkyl, or a group of the same general formula as Rxe2x80x21 as defined herein above; Rxe2x80x22 and Rxe2x80x23 are the same as Rxe2x80x21 or hydrogen, alkyl, alkenyl, amino- or hydroxy- or alkoxy-substituted alkyl, or substituted amino-substituted alkyl or alkenyl; or
b4) Rxe2x80x21 is an optionally substituted aryl group of general formula IV: 
xe2x80x83wherein Rxe2x80x25 is hydrogen or one or more substituents, and m is zero or an integer from 1 to 5; and Rxe2x80x22 and Rxe2x80x23 may be independently hydrogen, alkyl, amino- or hydroxy- or alkoxy-substituted alkyl, or substituted amino-substituted alkyl, or groups of the same general formula Rxe2x80x21.
Substitutions which are not explicitly specified are usual xe2x80x9cinertxe2x80x9d substituents, such as halogens, a hydroxyl group, alkyl having 1 to 4 carbon atoms, alkoxyl having 1 to 4 carbon atoms, acyloxyl having 1 to 4 carbon atoms and esterified carboxyl having 1 to 4 carbon atoms.
Advantageous examples of amines which form the salt with HMG-CoA reductase inhibitors are: (xc2x1)-1,2-dimethylpropylamine, 3-(2-aminoethylamino)-propylamine, n-butylamine, secondary butylamine, tertiary butylamine (TBA), dibutylamine, tertiary amylamine, cyclopentylarine, cyclohexylamine, cycloheptylamine, dicyclohexylamine (DCHA), N-methylcyclohexylamine, N,Nxe2x80x2-diisopropylethylenediamine (DIPEDA), N,Nxe2x80x2-diethylenediamine, N-methyl-1,3-propanediamine, N-methylethylenediamine, N,N,Nxe2x80x2,Nxe2x80x2-tetramethyl-1,2-diaminoethane, N,N,Nxe2x80x2,Nxe2x80x2-tetramethyl-1,4-diaminobutane, N,N,Nxe2x80x2,Nxe2x80x2-tetramethyl-1,6-diaminohexane, 1,2-dipiperidinethane, dipiperidinemethane, 2-amino-3, 3-dimethylbutane, N,N-dimethylcyclohexylamine, neopentylamine, adamantylamine, N,N-diethylcycohexylamine, N-isopropylcyclohexylamine, N-methyl-cyclohexylamine, cyclobutylamine and norborylamine. Preferably in terms of crystallization efficiency, combined with low toxicity and low costs, the amine is selected from the group consisting of n-butylamine, secondary butylamine, TBA, dibutylamine,tertiary amylamine, cyclohexylamine, DCHA, N-methylcyclohexylamine and DIPEDA. The amine may particularly be selected from-the group consisting of TBA, DIPEDA, DCHA and N-methylcyclohexylamine.
The amines specified above are advantageous over the direct isolation via the salts with ammonia in terms of purification efficiency. Furthermore, amines having a larger organic group, and especially those having bulky groups, generally show a more readily crystallization and to a lower extent form salts with unwanted side products when compared with amines having small organic groups. Accordingly, amines having at least one hydrocarbon residue with secondary or tertiary carbon atoms, or with cyclic hydrocarbon structure (either aromatic or aliphatic), and organic diamines are particularly suitable for the present invention.
Any type of HMG-CoA reductase inhibitors can be used according to the present invention. Those HMG-CoA reductase inhibitors selected from the group consisting of mevastatin, pravastatin, lovastatin, simvastatin, fluvastatin and atorvastatin have shown good results and are particularly preferred.
For isolating and purifying the desired HMG-CoA reductase inhibitor, the amine salt is most effectively formed directly from the crude medium of the respective HMG-CoA reductase inhibitor, which crude medium is usually derived from a fermentation broth as the result of a biotechnological process or from a reaction mixture as the result of a semisynthesis or the total synthesis and usually contains the desired HMG-CoA reductase inhibitor together with unwanted side products and impurities. The crude medium may preferably contain the HMG-CoA reductase inhibitor in its acid from, and the formation of the amine salt may be effected by simply adding the amine to the crude medium. The crude medium may be an organic phase or a mixture of an organic or an aqueous phase where the impure HMG-CoA reductase inhibitor is present in an organic solvent, such as ethyl acetate, ether or acetonitrile. Ethyl acetate is preferred as the organic solvent. After the biotechnological treatment, the crude medium in the organic phase is preferably obtained from the fermentation broth by a process including the step of extracting the HMG-CoA reductase inhibitor into the aforementioned organic solvent.
The process for the preparation of the amine salts of HMG-CoA reductase inhibitors may include the following steps:
a) contacting the medium containing the HMG-CoA reductase inhibitor, which is preferably in the acid form in an organic solvent, with at least one of the amines specified above,
b) optionally forming crystallization nuclei by known techniques,
c) filtering the crystals crystallized out,
d) washing the crystals with an organic solvent, and
e) drying the crystals.
The term xe2x80x9ccontactingxe2x80x9d includes the known techniques for the preparation of the salts from substances with acid properties and substances with alkaline properties. The crystallization is preferably carried out at a temperature between 0 and 30xc2x0 C., more preferably between 4 and 22xc2x0 C.
Since the amines specified above effectively form salts with the HMG-CoA reductase inhibitors, they are also particularly suitable as auxiliary materials or processing aids in a process for preparing the HMG-CoA reductase inhibitor in a purified form. Previously isolated HMG-CoA reductase inhibitors can be thus obtained in a higher purity. The purified form is usually prepared by crystallization. Accordingly, the present invention provides a process for the isolation and/or purification of a HMG-CoA reductase inhibitor.
In further aspects of the present invention, the salt as specified above is suitably used as the starting substance or the intermediate substance in a process for preparing the HMG-CoA reductase inhibitor which is in a modified form, in a pharmaceutically active salt form or in the lactone form. Specifically, the modified form is obtained by chemical modification or biotechnological modification, which modifications are known to those skilled in the art. The pharmaceutically active salt is preferably a metal salt, such as the sodium salt or the calcium salt.
In this connection, the term xe2x80x9ca process for the semi-synthetic preparation of HMG-CoA reductase inhibitorsxe2x80x9d means the preparation of HMG-CoA reductase inhibitors using any of the known chemical modifications of the HMG-CoA reductase inhibitors. An example of such process is the semisynthesis of simvastatin from lovastatin as the starting substance. Most preferably, the TBA salt of lovastatin is used as the starting substance.
Furthermore, the term xe2x80x9ca process for the biotechnological modification of HMG-CoA reductase inhibitorsxe2x80x9d means the preparation of HMG-CoA reductase inhibitors using microorganisms or enzymatic systems thereof for modifying HMG-CoA reductase inhibitors. An example of such process is the biotechnological conversion of mevastatin into pravastatin. The amine salt is preferably the TBA salt.
Furthermore, the term xe2x80x9ca process for the conversion of the amine salts of HMG-CoA reductase inhibitors into the pharmaceutically acceptable salts of HMG-CoA reductase inhibitorsxe2x80x9d includes processes for the preparation of HMG-CoA reductase inhibitors by one of the known methods wherein the amine salts of HMG-CoA reductase inhibitors are used as the starting substance. Specific examples of converted salts are the sodium salts of pravastatin and fluvastatin and the calcium salt of atorvastatin.
Furthermore, the term xe2x80x9cconversion of the amine salts of HMG-CoA reductase inhibitors into HMG-CoA reductase inhibitors in the lactone formxe2x80x9d includes processes for the preparation of HMG-CoA reductase inhibitors in the lactone form by one of the known methods wherein the amine salts of HMG-CoA reductase inhibitors are used as the starting substance. Examples of HMG-CoA reductase inhibitors converted into the lactone form are lovastatin, mevastatin or simvastatin.
The present invention is illustrated but in no way limited by the following examples.